Many pathological conditions, both congenital defects and acquired diseases, are associated with chromosomal aberrations such as amplifications, aneuploidy, potential breakpoints, insertions, inversions, deletions, duplications, rearrangements, and translocations. Moreover, pathogenic infections generally result in the presence of nucleic acid sequences of the infecting bacterium, virus, or fungus in the infected organism.
Establishing the presence or absence of a nucleotide sequence associated with a congenital defect, acquired disease, or infectious pathogen, through in vivo, in vitro, or in situ analysis of genomic DNA, chromosomes, chromosome fragments, or genes, can assist a clinician in reaching an appropriate diagnosis. For example, the expansion of a CGG trinucleotide repeat in the 5′UTR (UnTranslated Region) of mRNA of the fragile X mental retardation-1 (FMR1) gene allows the clinician to diagnose fragile X syndrome. This expansion leads to transcriptional silencing of the gene. However, other mechanisms, e.g. deletions of FMR1 and mutations, might also cause fragile X syndrome. The result, absence or reduced amounts of the gene product, FMRP, leading to the disease, is the same for both the expansion-caused silencing and for gene deletion. An example of a condition caused by a numerical anomaly is Down Syndrome, also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21, rather than two). Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome, an X. Other examples include Wolf-Hirschhorn syndrome, which is caused by partial deletion of the short arm of chromosome 4, and Jacobsen syndrome, also called the terminal 11 q deletion disorder. Some syndromes, such as Charcot-Marie-Tooth disease type 1A, may be caused by duplications, e.g., of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17. In other syndromes, such as Robertsonian translocation, an entire chromosome has attached to another at the centromere. Robertsonian translocations may only occur with chromosomes 13, 14, 15, 21 and 22 and the progeny of a heterozygous carrier of a Robertsonian translocation might, e.g. inherit an unbalanced trisomy 21, causing Down Syndrome.
Establishing the presence or absence of a nucleotide sequence associated with a congenital defect, acquired disease, or infectious pathogen, through in vivo, in vitro, or in situ analysis of genomic DNA, chromosomes, chromosome fragments, or genes, can also be invaluable to the clinician in selecting an appropriate course of treatment where a disease state has been diagnosed. For example, a breast cancer patient in whom the HER2 gene has been amplified may benefit from treatment with Herceptin™ (trastuzumab), a monoclonal antibody that recognizes HER2 protein. In another example, a clinician may choose to prescribe Erbitux® (cetuximab) or Vectibix™ (panitumumab) (therapeutic monoclonal antibodies that specifically recognize epidermal growth factor receptor (EGFR)) to a colorectal cancer patient in whom the EGFR gene is amplified.
Establishing the presence or absence of a nucleotide sequence associated with a congenital defect, acquired disease, or infectious pathogen, through in vivo, in vitro, or in situ analysis of genomic DNA, chromosomes, chromosome fragments, or genes, can also assist a clinician in providing a prognosis. Thus, breast cancer patients in whom the TOP2A gene is amplified or deleted have a worse prognosis than those in whom it is not.
Detecting the presence or absence of a nucleotide sequence generally entails recognition of the sequence by hybridization, or stabilization of a nucleotide double helix structure by hydrogen bonding between bases on opposite strands (A+T or G+C). In a basic example of hybridization, nucleic acid fragments or sequences bind to a complementary nucleic acid fragment or sequence. Detection by hybridization generally involves the use of nucleic acid probes designed to bind to, or “hybridize” with, a nucleic acid target such as, e.g., a DNA or RNA sequence.
Well known techniques exist in the art of molecular biology for detecting chromosome aberrations. So far, however, a fast, convenient, cheap, and user-friendly test which allows for widespread and routine detection of chromosome aberrations has not been available.
The efficiency and accuracy of nucleic acid hybridization assays depend primarily on at least one of three factors: a) denaturation (i.e., separation of, e.g., two nucleic acid strands) conditions, b) renaturation (i.e., re-annealing of, e.g., two nucleic acid strands) conditions, and c) post-hybridization washing conditions.
Traditional hybridization experiments, such as ISH assays, use a formamide-containing buffer to denature doubled stranded nucleic acid chains. Formamide is a solvent that has a destabilizing effect on the helical state of, for example, DNA, RNA, and analogs thereof, by displacing loosely and uniformly bound hydrate molecules. Furthermore, formamide stabilizes the coil state of DNA, RNA, and analogs thereof by ‘formamidation’ of the Watson-Crick binding sites of the bases.
The denaturation step is followed by the re-annealing of two complementary strands of nucleic acid chains, which is by far the most time-consuming aspect of an assay using hybridization. For example, in a traditional fluorescence in situ hybridization (FISH) protocol, re-annealing takes 14-24 hours, and can even take up to 72 hours. Examples of traditional hybridization times are shown in FIGS. 1 and 2.
Until now it was believed that the use of chaotropic agents, such as formamide (other chaotropic agents include guanidinium hydrogen and urea), which interfere with the Watson-Crick binding sites of nucleic acid bases and thereby disturb the hydrogen bonds between complementary nucleic acid bases, was the only way to lower the melting temperature (Tm) of the complementary chains, as is necessary for the denaturation step. However, although the use of chaotropic agents lowers the Tm, these agents appear to significantly prolong the hybridization time, as compared to hybridization in an aqueous solution without a chaotropic agent.
Formamide has disadvantages beyond a long processing time. Formamide is a toxic, hazardous material, subject to strict regulations for use and waste. Furthermore, the use of a high concentration of formamide appears to incur morphological destruction of cellular, nuclear, and/or chromosomal structure.
The aqueous compositions described herein allow the detection of nucleic acid sequences under conditions that have several potential advantages over the prior art, such as faster hybridization times, lower hybridization temperatures, and less toxic hybridization compositions.